Bone morphogenetic proteins (BMPs) are bone-inducing (osteogenic, osteoinductive) molecules that have been purified and characterized from bone (Sampath and Reddi, Proc. Natl. Acad. Sci. USA, 78: 7599 (1981)). The term “bone morphogenetic protein”, “BMP”, and “morphogen” are synonymous and refer to members of a particular subclass (i.e., the BMP family) of the transforming growth factor-β (TGF-β) superfamily of proteins (see, e.g., Hoffmann et al., Appl. Microbiol. Biotechnol, 57: 294-308 (2001); Reddi, J. Bone Joint Surg., 83-A(Supp. 1): S1-S6 (2001); U.S. Pat. Nos. 4,968,590; 5,011,691; 5,674,844; 6,333,312). All such BMPs have a signal peptide, prodomain, and a carboxy-terminal (mature) domain. The carboxy-terminal domain is the mature form of the BMP monomer and contains a highly conserved region characterized by seven cysteines that form a cysteine knot (see, Griffith et al., Proc. Natl. Acad. Sci. USA, 93: 878-883 (1996)). BMPs were originally isolated from mammalian bone using protein purification methods (see, e.g., Urist et al., Proc. Soc. Exp. Biol. Med., 173: 194-199 (1983); Urist et al., Proc. Natl. Acad. Sci. USA, 81: 371-375 (1987); Sampath et al., Proc. Natl. Acad. Sci. USA, 84: 7109-7113 (1987); U.S. Pat. No. 5,496,552). However, BMPs have also been detected in or isolated from a variety of other mammalian tissues and organs such as kidney, liver, lung, brain, muscle, teeth, and gut. Most BMPs (including BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, BMP-12, BMP-13) also stimulate cartilage and bone formation as demonstrated in a standard ectopic assay for bone formation (see, e.g., Sampath and Reddi, Proc. Natl. Acad. Sci. USA, 80: 6591-6595 (1983)). Accordingly, such authentic BMPs are also referred to as “osteogenic” even though they may also promote soft tissue regeneration.
The protein referred to routinely as BMP-1 is not an authentic member of the BMP family of osteogenic, tissue regenerative proteins. BMP-1 was originally isolated from highly purified BMP bovine bone extracts and was originally reported to induce the formation of cartilage in vivo in a subcutaneous (ectopic) bone formation assay (Wozney et al., Science, 242: 1528 (1988)). However, BMP-1 does not share significant amino acid sequence homology with other BMPs, nor does BMP-1 exhibit the characteristic signal peptide, prodomain, carboxy-terminal (mature domain), or cysteine knot found in other BMPs. In fact, BMP-1 was shown to be identical to procollagen C-proteinase, an enzyme essential for the proper assembly of collagen within the extracellular matrix (ECM) (Kessler et al., Science, 271: 360-362 (1996)). The erroneous status of BMP-1 within the TGF-β family resulted from flaws in the original bioassay for osteogenesis (Wozney et al., op. cit.) in which the cartilage observed in the bioassay appears to have been old growth plate cartilage contaminating the insoluble bone matrix that was misidentified as newly formed tissue (see, Reddi, Science, 271: 463 (1996)). As shown herein, unlike authentic osteogenic BMPs, the BMP-1-1 isoform does not induce cartilage or bone formation in a standard ectopic bone formation assay.
The BMP-1 gene is related to the Drosophila gene tolloid (TLD), which is implicated in the patterning controlled by the decapentaplegic (DPP) gene by virtue of its ability to activate TGF-β-like morphogens. The BMP-1 protein is now known to be an essential control point of morphogenesis during the cascade of pattern formation (Ge and Greenspan, Birth Defect Res., 78: 47-68 (2006)).
BMP-1 is the prototype of a small subgroup of metalloproteinases found in a broad range of species. In mammals, there are four BMP-1/TLD-related (or BMP-1/TLD-like) metalloproteinases. The gene encoding BMP-1 also encodes a second, longer proteinase that is encoded by alternatively spliced mRNA. With a domain structure that is essentially identical to TLD, this proteinase was designated mammalian Tolloid (mTLD) (Takahara et al., J. Biol. Chem., 269: 32572-32578 (1994)). In addition, there are two genetically distinct mammalian BMP-1/TLD-related proteinases, designated mammalian Tolloid-like 1 and 2 (mTLL1 and mTLL2). The prodomains of BMP-1/TLD-like proteinases must be proteolytically removed by subtilisin-like proprotein convertases (SPCs) (Leighton and Kadler, J. Biol. Chem., 278: 18478-18484 (2003)) to achieve full activity of these proteinases. The role of the prodomain of BMP-1/TLD-like proteinases appears to be in maintaining the BMP-1/TLD-like proteinases in a latent form (Marques et al., Cell, 91: 417-426 (1997); Sieron et al., Biochem., 39: 3231-3239 (2000); Leighton and Kadler, op. cit.).
BMP-1/TLD-related metalloproteinases are responsible for the proteolytic maturation of a number of extracellular proteins related to formation of the extracellular matrix (ECM). These include various collagens, small leucine-rich proteoglycans, SIBLING proteins, and the enzyme lysyl oxidases, laminin-5, and an anti-angiogenic factor from the basement membrane proteoglycan perlecan (Iozzo, Nat. Rev. Mol. Cell. Biol., 6: 646-656 (2005); Greenspan, Top. Curr. Chem., 247: 149-183 (2005); Ge and Greenspan Birth Defect Res., op. cit.). BMP-1 is also involved in releasing BMPs from extracellular matrix or in activating latent TGF-β family members, such as BMP-4, BMP-11 and GDF-8 (Wolfman et al., Proc. Natl. Acad. Sci. USA, 100: 15842-15846 (2003); Ge et al, Mol. Cell. Biol., 25: 5846-5858 (2005)).
The originally discovered form of BMP-1 is designated as BMP-1-1, and other BMP-1 isoforms encoded by splice variant RNA transcripts have been described on the transcriptional level and designated with sequential suffixes: BMP-1-2, BMP-1-3, BMP-1-4, BMP-1-5, BMP-1-6, and BMP-1-7 (Li et al., Proc. Natl. Acad. Sci. USA, 93: 5127-5131 (1996); Wozney et al., Science, 242:1528 (1988); Janitz et al., J. Mol. Med., 76:141 (1998); Takahara et al J. Biol. Chem., 269: 32572 (1994); Hillman et al., Genome Biol., 5: 16 (2004). As expected, the BMP-1 isoforms encoded by the splice variant transcripts share a number of domains, including leader peptide, proregion, and protease (catalytic) region. Only the original BMP-1, i.e., BMP-1-1, has previously been confirmed on the protein level following its isolation from bone. The sequences for BMP-1-2 and other BMP-1 isoforms were deduced from nucleotide sequences of the splice variant transcripts, but have not been described at the protein level.
Despite the correction in the literature of the identity of BMP-1-1, whether this protein or other BMP-1 isoforms have any role of therapeutic relevance remains to be elucidated.